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• • A—HUMAN NECESSITIES
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• This invention relates to the treatment of brain tumours using gene therapy.
• Background to the Invention The treatment of malignant glioma continues to challenge physicians and scientists.
• Thymidine kinase gene therapy using the Herpes Simplex virus thymidine kinase (HSVtk) gene, is one of the most promising treatment modalities, in attempts to change the survival of malignant glioma patients.
• HSVtk gene therapy is based on the ability of thymidine kinase to catalyze the phosphorylation of ganciclovir (GCV). Phosphorylated GCV acts as a toxic nucleotide analogue, leading to the death of the target cells.
• GCV Herpes Simplex virus thymidine kinase
• Retroviruses and adenoviruses have been used as vectors for gene therapy. Both vectors have certain advantages and limitations. Brain tumours are especially suitable for retrovirus-mediated gene transfer, since retroviruses can only infect proliferating cells while normal, non-dividing brain tissue remains intact. The gene transfer efficiency of retroviruses is relatively low, but could be improved by using retrovirus packaging cells instead of isolated viruses. The transduction time can theoretically be prolonged and the number of transfected cells increased.
• the transfected gene incorporates into the genome of the target cell and therefore long-term gene expression can be achieved.
• Summary of the Invention The present invention is based on the surprising finding that treatment of brain tumours using thymidine kinase gene therapy can be accomplished more effectively if an adenovirus is used as the vehicle to transfer the gene into tumour cells.
• an adenovirus comprises a gene encoding thymidine kinase, and medicaments containing it, are useful for treating a brain tumour.
• the tumour is treated following administration of gangciclovir or an equivalent compound.
• the thymidine kinase gene will typically be that derived from the Herpes Simplex virus (HSVtk).
• the adenovirus/thymidine kinase gene construct is shown to be more beneficial than retrovirus gene transfer. Description of the Invention
• a construct of the invention can be used to treat a tumour. Treatment may comprise the steps of:
• step (i) administering an adenovirus comprising a gene encoding a thymidine kinase, into the wall of the tumour cavity; and (ii) administering a compound that forms a cytotoxic compound when phosphorylated.
• the compound used in step (ii) may be ganciclovir or a derivative thereof. This method may be used to treat any tumour, preferably a brain tumour, e.g. a malignant glioma.
• the composition used in step (i) may be administered repeatedly, preferably in 40 to 80 separate applications.
• composition used in the present invention is preferably formulated without the addition of proteins (other than those associated with the adenovirus). This is believed to be preferable to those compositions where albumin is added to reduce the effects of degradative enzymes on the active components.
• this composition comprises glycerol as a stabiliser.
• the amount of active products that should be administered to a patient, in use of the invention can be determined by those skilled in the art, based on information provided herein and on the usual considerations such as the route of administration, the condition being treated and its status, etc.
• the following Example illustrates the invention.
• MRI magnetic resonance imaging
• a PA317/tk packaging cell line was prepared as described in Poptani et al, Cancer Gene Ther. 5:101-109 (1998). Briefly, 1.2 kb HSV1-TK cDNA (McKnight, Nucleic Acid Res. 8:5949-5964 (1980)) was subcloned into pLXSN retroviral plasmid (Miller et al, Mol. Cell. Biol., 5:2985-3902 (1986)) to create pLTKSN plasmid. Expression of the HSV1-TK is driven by 5' Moloney murine sarcoma virus LTR. The vector also contains an internal SV40 promoter which drives a neo ycin resistance (NEO) gene.
• NEO neo ycin resistance
• PA317 cell line was transfected with pLTKSN plasmid using calcium phosphate precipitation.
• PA317/3.0D5 cell line PA317/tk
• PA317/tk produced 106 cfu ml retroviruses as determined in 209F fibroblast assay (Yla-Herttuala et al, J. Clin. Invest. 95: 2692-2698 (1995)).
• packaging cells were expanded, trypsinized and diluted to 10 9 cells/10 ml Optimem (Gibco BRL). Cells were shown to be free of mycoplasma, other microbiological contaminants and wild-type viruses.
• ⁇ -galactosidase (lacZ)-containing BAG-retroviruses were produced in ⁇ CRIP. Titers of 6x10 s cfu/ml were produced and used as unconcentrated culture supernatant (DMEM, 0,5 % NCS, Gibco).
• DMEM unconcentrated culture supernatant
• Adenovirus In HSVtk adenovirus the expression cassette consisting of human cytomegalovirus (hCMV) enhancer and promoter element -HSV 1-TK cDNA -simian virus 40 (SV40) polyadenylation signal was subcloned into pAdenogal plasmid (Barr et al, Gene Ther. 1:51-58 (1994)) to create pAdCMVTK plasmid.
• Linearized pAdCMVTK and sub360 adenoviral DNA were cotransfected into 293 cells (ATCC CRL1573) and recombinant adenovirus, AdCMVTK, was generated through homologous recombination.
• AdCMVTK recombinant adenovirus
• the virus clone was purified by three rounds of plaque assay and after each round the presence of TK expression cassette in adenovirus genome was confirmed by PCR.
• Large-scale preparation of AdCMVTK was performed in 293 cells and the virus lysate was purified and concentrated in CsCl gradient, dialyzed and stored at -80 °C.
• Virus titer was determined by plaque assay in 293 cells. Adenovirus preparation was analyzed for integrity of TK expression cassette using restriction enzyme digestion followed by Southern blot analysis. The absence of wild-type replication-competent virus was confirmed by cytopathic assay on HeLa (ATCC CCL-2) and A549 (ATCC CC 185) cells. Virus preparation was also tested to be free from microbiological contaminants and lipopolysaccaride (Limulus assay, Sigma).
• adenovirus nuclear targeted ⁇ -galactosidase cDNA under a ⁇ -actin promoter and a CMV enhancer was cloned into El -deleted region of the adenoviral genome using homologous recombination.
• Adenoviruses were concentrated to titer 3 x 10 10 pfu/ml by ultracentrifugation. Purified virus was collected and dialysed with 5 mM Hepes (pH 7.8) and finally in 5 mM Hepes (pH 7.8) containing 20% glycerol.
• tumours in 14 patients were treated with HSVtk gene therapy.
• 7 control patients were transfected with control lacZ marker gene 4-5 days before resection. All patients had a Karnofsky score over 70. Mean age of the patients was 51 years (range 20-70 years). The tumour was recurrent in 13 cases (59 %). All patients received corticosteroids and antiepileptic medication and radiation therapy was used in de novo tumours. Operation and gene transfer
• tumour resection All patients underwent craniotomy and tumour resection.
• the resection was as radical as possible under the microscope.
• the diagnosis of malignant glioma was confirmed by frozen sections at the time of the operation.
• HSVtk retrovirus packaging cells (10 9 cells 10 ml) or adenoviruses (3xl0 10 pfu/lOml) were injected into the wall of the tumour cavity in 0.1-0.3 ml quantities, 10 mm deep, with 30-70 injections/patient.
• GCV treatment (5mg/kg/d) was delivered intravenously through the subclavian vein twice a day for 14 days.
• the medication started 14 days or 5 days after the tumour resection and gene transfer in retrovirus or adenovirus patients, respectively, ⁇ -galactosidase gene was transferred to seven control group patients via a catheter which was stereotactically inserted into the tumour. The catheter was left in place until the tumour was resected in craniotomy. Gene transfer vectors (BAG retroviruses, titer 6x10 s cfu, and adenoviruses, titer from 3xl0 8 to 3xl0 10 pfu) were injected into the tumour during three consecutive days, followed by tumour resection 1 -2 days later. Patients with ⁇ -galactosidase marker gene were not treated with GCV. All patients were treated according to standard clinical practice with radiation therapy. Magnetic Resonance Imaging
• HSVtk-treated patients were followed by MRI on first postoperative day, 4, 8 and 12 weeks after the gene transfer and every second month thereafter.
• tumour behaviour was graded as: progressive when there was even a slightest sign of tumour regrowth, stable when tumour status remained the same, and regressive when tumour volume decreased.
• Blood and urine samples were analyzed using routine methods before gene transfer, on the first postoperative day and weekly during hospitalization except for leucocyte differential count which was measured every second day during GCV medication.
• Anti-virus antibodies were measured before and two weeks after the gene transfer.
• PCR and wild-type virus assays were performed from plasma and urine samples before gene transfer and 3, 5, 7 and 21 days after the gene transfer.
• Neuropsychological testing was performed in order to determine cognitive functions and quality of life before operation and every second month after the treatment.
• WMS Wechsler Memory Scale
• Table 1 shows the results from the gene therapy experiments.
• Retrovirus packaging cells (PA317/tk) were used for seven patients and adenoviruses (Adv/tk) for seven patients.
• One patient (#) received repeated treatment with PA317/tk cells for two different tumours.
• Six of the cases were de novo tumours, others were recidives by previous operation (op), radiation therapy (rd) or chemotherapy (ch).
• Tumour was located in temporal (temp), occipital (occ), frontal (front), or parietal (pariet) lobe.
• (Sin) indicates the left side and (dx) the right side.
• Virus antibodies were measured from peripheral blood before and two weeks after the gene therapy.
• glioblastoma (gb) in 82% of the patients, two patients had anaplastic astrocytoma (aa) and two anaplastic oligodendroglioma (ao).
• Proliferation activity of the tumours was measured by ki67 immunohistochemical staining. Tumour identity was confirmed by glial fibrillary acid protein (GFAP) immunostaining. Outcome was measured in months and (*) indicates death of the patient. Difference between retro- and adeno-virally treated patients according to survival was significant (p ⁇ 0.05) by Fisher's exact test. The first postoperative MRI was done on 1 or 2 postoperative days after the tumour resection and gene therapy and the second postoperative MRI was done 3 months later.
• the resection was total if more than 98% of the tumour mass was resected, subtotal if resection was between 66-98% and partial if less than 66% of the tumour was resected.

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• 1998
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